Shikimic acid (trihydroxy-1-cyclohexene-1-carboxylic acid; Chemical Abstracts Registry Number 138-59-0) is the key precursor compound for the synthetic manufacture of oseltamivir ((3R,4R,5S)-4-(acetyl-amino)-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester; Chemical Abstracts Registry Number 196618-13-0) (Rohloff et al., 1998; Federspiel et al., 1999). Oseltamivir, an orally active inhibitor of the essential neuraminidase of influenza virus, was discovered by scientists at Gilead Sciences Inc. of Foster City, Calif. (Kim et al., 1997; Kim et al., 1998; Abrecht et al., 2004; Bischofberger et al., U.S. Pat. No. 5,763,483; Lew et al., U.S. Pat. No. 5,866,601; Bischofberger et al., U.S. Pat. No. 5,952,375; Bischofberger et al., European Patent EP 0759917 B1; Bischofberger et al., European Patent EP 0976734 B1).
During influenza virus replication, new virus particles are bound to a sialic acid side-chain on the virus receptor protein. The mechanism of the viral neuraminidase is to cleave off this sialic acid and release the newly replicated virus particles. Oseltamivir is a sialic acid analog that inhibits this cleavage reaction by binding to the active site of the neuraminidase. These abortively infected cells are destroyed, stopping the spread of the virus within the host. Oseltamivir has potential use in influenza pandemics, including of “bird flu”, in the form of the pharmaceutical TAMIFLU (De Clercq, 2002; Bradley, 2005; Farina and Brown, 2006). The pharmaceutical TAMIFLU is the phosphate salt of oseltamivir (Chemical Abstracts Registry Number 204255-11-8; also known as Roche compound Ro 64-0796/002 and Gilead Sciences compound GS-4104-02). TAMIFLU was first marketed by Roche in October 1999 (Farina and Brown, 2006). However, the large-scale production of the drug has been limited by the low availability of the shikimic acid precursor material.
Shikimic acid is a scarce and expensive chemical substance, being obtained principally from the seeds of woody shrubs, namely the Chinese star anise shrub (Illicium verum) native to China, and the shikimi-no-ki shrub (Illicium anisatum, formerly called Illicium religiosum) from whence shikimic acid got its name, native to Japan (Haslam, 1974; Sadaka and Garcia, 1999; Payne and Edmonds, 2005). About 30 kilograms of star anise or shikimi-no-ki seeds are required to produce one kilogram of shikimic acid (Farina and Brown, 2006). However, this natural source is limited, and insufficient to meet worldwide demand for TAMIFLU (Bradley, 2005).
1.3 grams of shikimic acid are required to manufacture the 10 doses of TAMIFLU needed to treat one person (Bradley, 2005). Production of a supply of TAMIFLU sufficient for treating 400 million people (a conservative estimate of the need in the event of an influenza pandemic) would require 520,000 kilograms of shikimic acid. Worldwide annual production of shikimic acid is currently only about 100,000 kilograms. Another estimate of the need for TAMIFLU in the event of a severe influenza pandemic is 30 billion doses, requiring 3.9 million kilograms of shikimic acid (Bradley, 2005).
Various approaches to resolving the problem of shikimic acid scarcity have been recently explored. One is the production of shikimic acid by microorganisms by a fermentation-based process (Farina and Brown, 2006). A fermentation-based process is described in Bogosian et al., International Patent Application No. PCT/US2008/060079, the entire contents of which are incorporated herein by reference for all relevant purposes and described below as the “fermentation method.” Bogosian et al. includes a survey of fermentation-based processes, including citation of many related references, many of which are listed in the “REFERENCES” section elsewhere herein. Another method for production of shikimic acid is based on new chemical synthesis routes to oseltamivir phosphate that do not utilize scarce natural products as precursor compounds, but rather use inexpensive and widely available chemicals (Fukuta et al., 2006; Yeung et al., 2006). The chemical routes that have been developed to date are functional only as academic, bench-scale syntheses, and are not efficient industrial processes that could compete with the current shikimic acid-based manufacturing process for oseltamivir phosphate (Farina and Brown, 2006). Still other methods utilize plants for production of shikimic acid (as detailed below).
To understand the fermentation-based processes and other processes for production of shikimic acid, it would be useful to briefly review the biosynthetic pathway to shikimic acid. This pathway is known both as the common aromatic biosynthetic pathway (Herrmann, 1983; Pittard, 1987; Pittard, 1996) because it leads to (among other things) the aromatic amino acids, and also as the shikimate pathway (Haslam, 1974) after the metabolic intermediate in the pathway that was identified first. Several entire books and comprehensive review articles have been devoted to this important metabolic pathway (Haslam, 1974; Weiss and Edwards, 1980; Herrmann, 1983; Conn, 1986; Pittard, 1987; Haslam, 1993; Herrmann, 1995a; Herrmann, 1995b; Pittard, 1996; Herrmann and Weaver, 1999; Bongaerts et al., 2001; Kramer et al., 2003).
The common aromatic biosynthetic pathway is present in plants, bacteria, fungi, and other eukaryotic microorganisms. A search of on-line databases, specifically PubMed and the National Center for Biotechnology Information (NCBI), indicated that in addition to plants, bacteria, and fungi, the pathway is present in Stramenopiles such as brown algae and diatoms, Alveolata (within the Protista kingdom) such as ciliates, dinoflagellates and apicomplexa parasites, and various Euglenozoa. The common aromatic biosynthetic pathway in the bacterium Escherichia coli is shown in FIG. 1. The common aromatic biosynthetic pathway is not found in most higher animals, such as nematodes, insects and other arthropods, mollusks, and vertebrates and other chordates including fishes, amphibians, reptiles, birds and mammals. It has been established by others that the common aromatic biosynthetic pathway is present in the parasitic protozoan microorganisms known as apicomplexa (Roberts et al., 1998; McConkey, 1999; Roberts et al., 2002). It has also been suggested by others that the common aromatic biosynthetic pathway may be present in some higher animals, specifically in basal metazoans among the marine and freshwater invertebrates known as cnidarians (or coelenterates), including corals, sea anemones, jellyfishes, and hydroids (Starcevic et al., 2008). It is to be understood that statements that the common aromatic biosynthetic pathway is present in microorganisms mean that the pathway occurs in microscopic organisms and taxonomically related macroscopic organisms within the categories algae, Archaea, bacteria, fungi, and protozoa; this includes prokaryotes, including cyanobacteria, as well as unicellular eukaryotic organisms.
The fact that microorganisms possess the common aromatic biosynthetic pathway, and depend on it for the biosynthesis of many essential cellular components, and that mammals (including humans) lack the pathway, make the enzymes of the pathway attractive targets for new classes of antimicrobial therapeutic agents (Davies et al., 1994; Roberts et al., 1998). Any such therapeutic agents that are based on shikimic acid would increase the demand for shikimic acid. Indeed, 6-fluoroshikimic acid has been found to be an effective anti-bacterial compound (Davies et al., 1994; Bornemann et al., 1995) and anti-parasitical compound (McConkey, 1999; Roberts et al., 2002). Shikimic acid has also been converted into compounds that exhibited a significant inhibitory effect on cell proliferation, opening their possible use as anti-cancer chemotherapeutic agents (Tan et al., 1999). Thus, shikimic acid could serve as an important building block for a wide array of important classes of drugs, including anti-viral, anti-bacterial, anti-parasitical, and anti-cancer drugs.
As noted, other methods for production of shikimic acid utilize plants. In some plants and plant tissues, such as seeds, shikimic acid naturally accumulates to high levels. Chinese star anise and various species of evergreen trees are examples of these. Shikimic acid does not normally accumulate to detectable levels in, for example, vegetative tissues of important agronomic crops such as alfalfa, soybean, wheat, corn, sugar beet, etc.
Shikimic acid is a cyclitolcarboxylic acid, a class of compounds that also includes quinic acid. Quinic acid (1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid; Chemical Abstracts Registry Number 77-95-2) is also a starting material for the synthesis of various biologically important molecules, including oseltamivir. It is utilized in the synthesis of FK-506, an immune suppressive agent useful in preventing organ transplant rejection (Rao et al., 1991) and many natural products that are otherwise difficult to obtain, e.g., mycosporin (White et al., 1989) and D-myo-inositol-1,4,5-triphosphate (Falck et al., 1989). Quinic acid can be converted to shikimic acid (Dangschat et al., 1938 & 1950; and Adachi et al., 2006) and also to the methyl ester of shikimic acid (Cleophax et al., 1971 & 1973). Quinic acid is found in cinchona bark, coffee beans and the leaves of certain plants, and is made synthetically by hydrolysis of chlorogenic acid.
Glyphosate is well-known as a highly effective and commercially important herbicide useful for combating the presence of a wide variety of unwanted vegetation, including agricultural weeds (for example U.S. Pat. Nos. 3,799,758 and 4,405,531). Glyphosate and its various salts are essentially nonselective, meaning that when applied post-emergence they control a wide variety of annual and perennial weeds.
Glyphosate is a xylem and phloem mobile herbicide. Once absorbed by treated foliage, glyphosate is mobilized from treated leaves to other plant parts, such as roots or newly formed leaves. Mobilization from treated leaves will ultimately be limited by a direct herbicide effect on the treated leaves. When the herbicide effect is greater, the rate and extent of mobilization will be decreased. This effect on mobilization is greater with increased concentration of herbicide treatment, or when the environmental conditions at treatment are particularly stressful, such as under extreme dry, hot or cold conditions.
The mode of action of glyphosate is inhibition of the common aromatic biosynthetic pathway by inhibition of the chloroplast enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Inhibition of EPSPS by glyphosate is non-reversible and treated plants are eventually “starved” of the essential end-products of the common aromatic biosynthetic pathway. Since this affects new growth to a greater extent than existing parts of the plant, the symptoms of glyphosate treatment appear to be slow to develop.
Shikimic acid is a natural product that does not normally accumulate to high levels in plants. However, when susceptible plants are treated with glyphosate, they accumulate shikimic acid (and also probably accumulate smaller amounts of the unstable metabolic intermediate 5-phosphoshikimic acid) to high levels. It is possible then, to use this system to produce large quantities of shikimic acid in any plant which is susceptible to glyphosate, both in plants which do not normally accumulate shikimic acid, and in plants which may naturally accumulate shikimic acid to some level. Further, if this method is applied to a normal agronomic crop system, including but not limited to soybean, alfalfa, corn, and wheat, it can be used to produce shikimic acid to industrial-scale levels and could potentially alleviate the limitation of shikimic acid in the production of products such as the neuraminidase inhibitor TAMIFLU.
Recently, with the bioengineering of glyphosate-resistant crops such as soybean, corn, cotton, canola, alfalfa, and sugar beet, glyphosate use has increased. When glyphosate is applied to glyphosate-resistant plants, no visual or biochemical injury symptoms are detected; however, any susceptible, undesirable plants (such as weeds) that may be present are controlled. The crops are still harvested in the conventional manner. For example in ROUNDUP READY Soybeans, the bean pods are harvested, and in ROUNDUP READY Cotton, the cotton bolls are collected. It is not typical to harvest the entire plant in such crops.
Previous research has identified shikimic acid accumulation in plants as a consequence of contacting a susceptible plant with glyphosate (see, for example, Amrhein et al., 1980; Harring et al., 1998; Pline et al., 2002; Mueller et al., 2003; Buehring et al., 2007; and Henry et al., 2007). Anderson et al. (2001) disclose a method for the determination of shikimic acid in plant tissue after exposure of the plant to glyphosate. Shikimic acid analysis of the plant tissue was performed using water extraction followed by high-performance liquid chromatography (HPLC) analysis. Anderson International Publication No. WO 2008/027570 A2 describes a method of isolating shikimic acid from a plant (wheat) that has been treated with glyphosate to increase the amount of shikimic acid in the plant.
Despite the knowledge of shikimic acid being present in plants treated with glyphosate, substantial efforts by others to develop new sources of shikimic acid have failed even though there has been a significant awareness and long-felt need for improved sources of shikimic acid, based on highly publicized concerns related to global pandemics of “bird flu” and other influenza-type viruses.
The research exemplified herein has shown that treatment of susceptible plants with glyphosate can also result in the accumulation of quinic acid. This accumulation of quinic acid provides a new source of quinic acid. It is possible then to treat susceptible plants with glyphosate to produce quinic acid on industrial-scale levels. The resulting quinic acid can be used in the production of bioactive chemical compounds such as FK-506 and TAMIFLU. Alternatively, the resulting quinic acid can be used to prepare shikimic acid which in turn may be used in the production of products such as TAMIFLU.
One object of the present invention is development of processes for production of shikimic acid and quinic acid utilizing plants. Another object of the present invention is development of processes for recovery of shikimic acid, regardless of the manner of its production (e.g., by the “plant method” or “fermentation method” as detailed elsewhere herein).